Dental caries and pulpitis cause developmental arrest of tooth roots of permanent teeth in children and adolescents. Wide-open root apex is susceptible to trauma and infections, frequently resulting in tooth loss at a young age (typically <18 years old). Tooth loss in children and adolescent patients is devastating because titanium dental implants are contra-indicated due to metal submerge in the alveolar bone that undergoes active vertical growth. Currently, there is no effective clinical treatment for infected permanent teeth with open root apex in children and adolescent patients. Regenerative cells or cues that promote dentin formation and continuous root development offer the only hope to address this unmet clinical need. Craniofacial mesenchymal stem/progenitor cells in development differentiate into odontoblasts that form dentin. Our preliminary data show that a panel of 948 genes and transcriptional factors are up- or down-regulated when craniofacial mesenchymal stem/progenitor cells differentiate into odontoblasts. We delivered two rationally selected factors in endodontically treated root canals in a preclinical model in vivo and found robust dentin regeneration in the form of tubular dentin with dentinal tubules and odontoblast processes as demonstrated by electron microscopy. Mechanical properties of the regenerated dentin were at par with native dentin that served as a substrate for the newly formed, regenerated dentin. Strikingly, no cells were transplanted in endodontically treated root canals. Thus, all of the regenerated dentin was derived from host endogenous cells that were recruited by these two molecular cues. In this application, we transform our newly gained knowledge on endogenous dentin regeneration in mature teeth into immature teeth with wide-open root apex in a preclinical, large animal model that is equivalent to developing permanent teeth in children and adolescent patients. This translation from mature teeth to immature teeth is far from a simple chronological switch, but will address two scientifically unknown and clinically relevant questions: 1) Can single molecular cues not only induce the recruitment of sufficient endogenous apical papilla stem/progenitor cells, but also transform them into dentin-generating odontoblasts? 2) Can spatiotemporal delivery of molecular cues promote directional dentin growth to complete root development? Accordingly, the overall objectives of this proposal, in response to PAR-13-137 (Bioengineering Research Grants, BRG), are to develop novel strategies for dentin regeneration in immature teeth with wide open root apex in a preclinical, large animal model. Our overarching hypothesis is that dentin regeneration in immature teeth with wide-open root apex is enabled by signal gradients of novel molecular cues that induce the recruitment and differentiation of endogenous stem/progenitor cells.